REVIEW Open Access

The production of alpha/beta and gamma/delta double negative (DN) T-cells and theirrole in the maintenance of pregnancyJohn C. Chapman*, Fae M. Chapman† and Sandra D. Michael†

Abstract

The ability of the thymus gland to convert bone marrow-derived progenitor cells into single positive (SP)T-cells is well known. In this review we present evidence that the thymus, in addition to producing SPT-cells, also has a pathway for the production of double negative (DN) T-cells. The existence of this pathway wasnoted during our examination of relevant literature to determine the cause of sex steroid-induced thymocyte loss.In conducting this search our objective was to answer the question of whether thymocyte loss is the end productof a typical interaction between the reproductive and immune systems, or evidence that the two systems areincompatible. We can now report that “thymocyte loss” is a normal process that occurs during the production ofDN T-cells. The DN T-cell pathway is unique in that it is mediated by thymic mast cells, and becomes functionalfollowing puberty. Sex steroids initiate the development of the pathway by binding to an estrogen receptor alphalocated in the outer membrane of the mast cells, causing their activation. This results in their uptake of extracellularcalcium, and the production and subsequent release of histamine and serotonin. Lymphatic vessels, located in thesubcapsular region of the thymus, respond to the two vasodilators by undergoing a substantial and preferentialuptake of gamma/delta and alpha/beta DN T- cells. These T- cells exit the thymus via efferent lymphatic vessels andenter the lymphatic system.The DN pathway is responsible for the production of three subsets of gamma/delta DN T-cells and one subset of alpha/beta DN T-cells. In postpubertal animals approximately 35 % of total thymocytes exit the thymus as DN T-cells, regardlessof sex. In pregnant females, their levels undergo a dramatic increase. Gamma/delta DN T-cells produce cytokines that areessential for the maintenance of pregnancy.

Keywords: Mast cells, Sex steroids, DN pathway, DN T-cells

BackgroundSteroids play a commanding role in all aspects ofreproduction [1]. They do this through the mediation ofsteroid receptors, a process that is purported to involvecomponents of the immune system [2, 3]. However, re-search conducted during the development of oral con-traceptives suggests that a ligand-receptor interactionbetween the two systems may not be possible. This be-came apparent when it was found that injecting femalerats with estrogen and testosterone caused the thymus

to suffer a severe loss of thymocytes and to undergothymic involution [4]. Although this finding wasregarded as atypical and due to exposing the thymus toexcessive levels of the two steroids [4], a more recent re-port found that physiological levels of estrogen alsocause thymocyte loss and thymic involution [5]. Takenin toto, these studies have led to the theory that sex ste-roids initiate, and then perpetuate the aging process ofthe immune system [6]. This would suggest that the twosystems are ill-suited for each other. We disagree withthis premise and will present evidence to show thatthymocyte loss, instead of being due to incompatibility,results from a sex steroid-induced release of γδ and αβdouble-negative [DN] T- cells into the lymphatic system.

* Correspondence: johnchapman1@juno.com†Equal contributorsDepartment of Biological Sciences, Binghamton University, Binghamton, NewYork 13902-6000, USA

© 2015 Chapman et al. This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in anymedium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 DOI 10.1186/s12958-015-0073-5

In brief, the discharge of these T-cells occurs when sexsteroids bind to the estrogen receptor alpha [7] ofthymic mast cells. Mast cell activation, coincident with arapid influx of extracellular calcium, results in the re-lease of vasodilators such as histamine and serotonin [8].Nearby lymphatic vessels become enlarged and undergoa preferential and significant uptake of the aforemen-tioned DN T- cells. The T-cells then exit the thymus viaefferent lymphatic vessels and enter the lymphatic sys-tem. These DN T-cells play a key role in the mainten-ance of pregnancy.

ReviewAfter exposure to hydrocortisone and dexamethasone,thymocytes become apoptotic and undergo cell death[9, 10]. Whether or not sex steroids cause thymocyteloss by apoptosis was examined in a number of stud-ies in which animals were subjected to estrogen ad-ministration. Unfortunately, the results were notablefor their lack of consensus. Estrogen treatment insome studies resulted in an increase in the rate ofthymocyte apoptosis [11–13], whereas in other re-ports, estrogen treatment produced little or no evi-dence of apoptotic death [14, 15]. In a further studyof the phenonomen, Zoller et al. [5] found that preg-nant mice undergo extensive thymocyte loss andthymic involution without thymocyte apoptosis evertaking place. In pregnant mice, the levels of estrogenrange between 7 ng/ml to 13 ng/ml [16]. Studies thatreported a high incidence of thymocyte apoptosisinjected the animals with levels of estrogen far in ex-cess of these values [11–13]. Thus, without evidenceto show that physiological levels of estrogen causeapoptosis, this process can be ruled out as the reasonfor thymic involution and thymocyte loss.Some investigators have proposed that thymocyte loss

takes place because estrogen blocks T-cell production atthe precursor level. This premise came from a study inwhich estrogen treatment resulted in an increase in thelevels of the earliest CD44+ progenitors and a depletionof all defined thymocyte subsets of CD4+ and CD8+ T-cells [17]. Other researchers have proposed that thymicinvolution is due to an estrogen-induced reduction inearly thymic progenitors [15]. These studies suggest thepossibility that thymocyte loss is the result of an alter-ation in T-cell production.Martin et al. [18, 19], using light and electron micros-

copy, observed an estrogen-induced loss of thymocytesin the subcapsular and deep cortex of the rat thymus. Inthe medullary region, they found evidence of an increasein the vascular permeability of blood vessels located nearthe corticomedullary junction. Lymphocytes were oftenseen migrating through the enlarged walls of these bloodvessels. They concluded that “the release of lymphocytes

from the thymus seems to be the main factor inducingthymic involution.” Others have observed that thelymphatic vessels in involuted thymuses are packed withlymphocytes (T-cells) [20, 21].Although not identified as such, these lymphatic ves-

sels would have to be efferent lymphatic vessels, sincethe thymus lacks the afferent variety [22, 23], an import-ant distinction.Oner and Ozan [24] reported that prolonged treat-

ment of female rats with either testosterone or estrogen(daily for 3 weeks) caused extensive thymic involution.This involution was accompanied by a loss of thymo-cytes in the subcapsular region as well as in the deepcortex. Blood vessels in the thymic medulla were also en-larged, as was noted in the report by Martin et al. [18, 19].The most significant finding by Oner and Ozan, however,was the identification of mast cells in connective tissue ofthe thymic capsule and in the stroma of the thymic me-dulla. In untreated control rats, mast cells were sparselydistributed, whereas in steroid-treated animals, they wereincreased in number and often found in clumps. The factthat mast cells secrete vasodilators leaves little doubt as tothe cause of the increase in vascular permeability; whichmay be the reason why involuted thymuses were packedwith lymphocytes [20, 21]. As to the identity of these lym-phocytes, studies of estrogen-injected [25, 26] and thymic-implanted nude mice [27] revealed that “thymocyte loss”was the result of the discharge of two subsets of DN T-cells [25, 26]. One subset had a typical αβ T-cell receptor(TCR), and the other had a unique γδ TCR.

T-cell productionThe thymus gland consists of two distinct lobes, eachcomposed of a central medulla and an outer cortex. Twolayers of connective tissue, separated by a sinus, encap-sulate both lobes. In most species, the capsule gives riseto trabeculae that penetrate the cortex and terminate atthe corticomedullary junction, thereby providing a struc-tural link to the medulla. A basement membrane sup-ports a specialized flattened epithelium lining thesubcapsule and trabeculae. Arteries travel within thecapsule and then either enter the cortex as arterioles orcontinue within the trabeculae until they reach the corti-comedullary junction, where they pass into the medulla.Arterioles become progressively smaller and continuethroughout the thymus as capillaries, undergoing even-tual transformation into venous capillaries and subse-quent enlargement to form postcapillary venules (PCVs).These venules ultimately lead to major blood vessels thattravel back to the trabeculae, where they leave in closeproximity to the incoming arteries [21, 22, 24, 28–30].The distribution of blood and lymphatic vessels (LVs)

is not uniform. For example, the cortex lacks PCVs,whereas the medulla contains a large number [21, 22].

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 2 of 14

In addition, the cortex contains a small contingent ofbranched LVs, located mainly in the subcapsular region[31]. These vessels extend into the capsule and extralob-ular region and connect to efferent lymphatic vessels(ELVs) [21, 22, 31]. In the medulla, LVs are more plenti-ful and are localized in the region of the corticomedul-lary junction. These connect with ELVs in thetrabeculae. Mast cells are absent from the cortex, butare found nearby in the connective tissue of the capsule[24]. In the medulla, mast cells are located in proximityto both LVs and PCVs [24, 32, 33]. Notably, in the invo-luted thymus the number of mast cells is significantly in-creased [24, 34, 35].T-cell progenitors produced in the bone marrow reach

the thymus via the arterial branch of the circulatory sys-tem. Upon entering the gland, they travel through arteri-oles as well as arterial and venous capillaries until theyarrive at the PCVs. The progenitors then pass into thethymic stroma, using a process referred to as extravasa-tion, or diapedesis. Diapedesis takes place in vessels thathave walls of endothelium and lack a muscular layer[36], such as PCVs and LVs. Endothelial cells are uniquein that lymphocytes are able to insinuate themselves be-tween cellular junctions, then pass either into, or out of,the thymic stroma [37–39]. Lymphocyte movement isaided by estrogen-activated mast cells through their pro-duction of histamine and serotonin, which in turn,causes a widening of the cellular junctions of the endo-thelial cells [36]. Diapedesis in PCVs is unidirectionaland limited to lymphocyte movement from the lumeninto the thymus [40, 41]. For passage out of the thymus,the T-cells utilize LVs [42–46], since these are capable ofreverse diapedesis [47, 48].Figures 1 and 2 are graphic representations of thymo-

cyte development in pre- and postpubertal mice. Shownin each figure are four spatially defined developmentalstages in the cortex that Lind and colleagues [49] havemapped using the progenitor markers, CD117 andCD25. Differential expression of these two markers re-flects developmental changes in the thymocytes as theymove outward from the corticomedullary junction intothe cortex. In this process, thymocyte movement is aidedin large part by an interaction between chemokines pro-duced by cortical epithelial cells in specific areas of thecortex, and thymocyte chemokine receptors [50]. Stage 1(CD117+CD25—) begins at the corticomedullary junctionand is characterized by thymocytes with multilineage po-tential. These cells, in addition to giving rise to T lym-phocytes, can also evolve into B lymphocytes, as well asdendritic and NK cells. Cells that reach stage 2 (CD117+CD25+) no longer have the ability to become B lympho-cytes and NK cells, but can give rise to αβ T-cells, γδ T-cells, and dendritic cells. Intracellular CD3ε protein isdetected at this stage [51]. In addition, a significant

amount of thymocyte proliferation occurs at stage 2.Cells that reach stage 3 (CD117—CD25+) are committedto T-cell lineage. Intracellular CD3ε protein synthesiscontinues unabated. TCR β protein is first detected atthis stage. Cells that express productive rearrangementsof TCR β with an α chain are selected to proliferate andproceed to stage 4, a process termed as β selection. Atstage 4 (CD117—CD25—), thymocytes have reached thesubcapsular region of the cortex with their TCR in placeand γ and δ binding components added to the CD3complex. Most have traversed the αβ TCR developmentalpathway and are characterized as αβ CD4—CD8— double-negative (DN) T-cells. Thymocytes that have developed aγδ TCR are referred to as γδ DN T-cells. Their numberscomprise 5-10 % of total DN T-cells [27, 52].

DN T-cell pathwayGamma/delta T-cells are not found in the thymus be-yond stage 4 of development [51]. This suggests: 1) anabsence of thymic tissue specifically dedicated to thecontinuation of their chemokine-facilitated travel; and 2)a strong probability that they leave the thymus directlyafter they are produced. Lymphatic vessels locatednearby in the subcapsular cortex are very likely theirmeans of exit. In mice, the DN pathway is operationalshortly shortly after birth, with DN T-cells being foundin the liver and spleen of 4-day-old animals [52, 53].Notably, the levels of αβ DN T-cells exceed that of γδDN T-cells by a factor of 4:1. Shown in Fig. 1 are theproposed exit pathways of γδ DN T-cells and αβ DN T-cells in prepubertal mice. As is indicated, most T-cellsleave the thymus via ELVs located in the medulla (solidblack arrows). However, in postpubertal mice (Fig. 2) alarge number of γδ DN T-cells and αβ DN T-cells exitthe thymus via ELVs located in the subcapsular cortex(solid red arrows) as the result of a sex steroid-inducedactivation of thymic mast cells.Estrogen activation of mast cells takes place via a

membrane-associated (non-genomic) estrogen receptor-α (ER-α) [7]. This activation results in an influx of extra-cellular calcium and the synthesis and release of gran-ules of histamine and serotonin [8]. Mast cell activationcan be achieved with concentrations of estrogen between10−11 M and 10−9 M (2.7 pg/ml to 270 pg/ml) [54]. Tes-tosterone activation requires levels that are 10 times thatof estrogen [55]. Activation by the weak androgen, dehy-droepiandrosterone (DHEA), necessitates levels that are1000 times that of estrogen [56, 57]. Dihydrotestosterone(DHT) is also a mast cell activator [58]. Progesterone isan inhibitor of estrogen activation [59].In postpubertal animals, endogenous sex steroids at-

tain levels that are fully capable of activating thymicmast cells. For example, circulating levels of testosteronein male mice and rats average 18.7 ng/ml and 5.8 ng/ml,

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 3 of 14

Fig. 1 (See legend on next page.)

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 4 of 14

respectively [60]. In nonpregnant female mice and rats,the levels of estrogen are 66 pg/ml and 30.6 pg/ml, re-spectively [61, 62], and in pregnant mice, estrogen levelsrange from 7 ng/ml to 13 ng/ml [16]. Strong evidencethat the ER-α plays a role in estrogen-induced thymic in-volution is indicated by studies of estrogen receptorknockout mice (ERKO). In these animals, the ER-α isnonfunctional; consequently, the thymus undergoes onlyminimal estrogen-induced involution [63, 64].

Classic T-cell pathwayIn contrast to the fate of γδ DN T-cells, αβ DN T cellsretain the option of continuing their development in thethymus. This choice is exercised when CD4 and CD8markers are expressed, and αβ DN T-cells become CD4+

CD8+ double-positive (DP) T-cells. In utilizing this op-tion, DP T-cells apparently lose the ability to access theDN pathway. This is either because they are restrictedfrom doing so or have left the area of the subcapsularLVs. Abo’s group reports a total absence of DP T-cells inthe pool of SP T-cells and DN T-cells found in the liverof estrogen-injected mice [25]. In the next developmen-tal stage, DP T-cells undergo positive selection, a pro-cedure concurrent with the production of two subsets ofsingle-positive (SP) MHC restricted T-cells. These sub-sets are CD4+ (class II MHC-restricted) and CD8+ (classI-MHC restricted) T-cells, and as such, they continue oninto the medulla. Here they undergo negative deletion, aprocess in which their αβ TCRs are exposed to ectopicself-antigens. Production of these antigens is under thedirection of the autoimmune regulator (Aire) promotor[65]. Fully mature CD4+ Helper, CD4

+ CD25+ Foxp3+Regu-latory, and CD8+ Cytotoxic T-cells exit the thymus via LVslocated in the medulla (Fig. 1, solid black arrows; Fig. 2,dashed red arrows).

Interaction between DN and SP pathwaysIt should be noted that the permeability of all LVsand PCVs is increased through the combined actionof sex steroids and mast cells. This results in an in-creased entry of T-cell progenitors and an enhance-ment in the exit rate of DN T-cells. To gain anappreciation of the levels of thymocytes that exit thethymus via the DN pathway, one only has to meas-ure the total number of thymocytes prior to, andafter castration. Fortunately, this has been done by anumber of researchers. For example, Pesic et al. [66]

reported that thymocyte levels in castrate and intact60-day-old Albino-Oxford male rats were 1050 × 106

and 650 × 106, respectively. This would suggest thatmast cell activation has facilitated the exit of 38 %of total thymocytes. Notably, these thymocytes werereported to originate from the cortex. In a study ofintact and castrated 60-day-old female Sprague–Dawley rats [57], the results indicated that estrogencaused 44 % of total thymocytes to exit via the DNpathway. Findings from a third study of male and fe-male adult Wistar-albino rats [24] revealed that tes-tosterone and estrogen affected a reduction of 31 %and 30 % of total thymocytes, respectively. Thesestudies demonstrate the effect of sex steroids in al-tering the dynamics of T-cell production. In the cas-trate animal, the thymus produces mainly SP T-cells.Their production time takes 3–5 days in the cortexand 12–16 days in the medulla [67], for a total of~21 days. In the intact animal, a significant numberof DN T-cells exit the thymus via the DN pathway. Theirtotal production time is 3–5 days. In these animals, the re-ports of a reduction in thymocyte levels of ~ 35 % [24, 57,66] strongly indicates that progenitor replacement doesnot keep pace with DN T-cell production.Pesic et al. [66] also measured thymocyte levels in the

cortex and medulla of intact and castrate male rats.With this information we were able to examine theeffect of the discharge of DN T-cells in altering thelevels of SP T-cells. For example, in the castrate ani-mal (Fig. 3) a comparison between thymocyte levelsin the cortex and medulla indicates that 2 % of totalthymocytes leave via the DN pathway, and 11 % reachthe medulla to become SP T-cells. Without castration(Fig. 4), a similar comparison suggests that 38 % oftotal thymocytes exit via the DN pathway and only7 % reach the medulla. Thus, the production of DNT-cells is the result of a proverbial “fork in the road”of T-cell development. Thymocytes can either leavethe thymus as DN T-cells, or they can remain in theclassic T-cell pathway and exit as SP T-cells. Theirpathway of development is determined by sex ste-roids. For example, during pregnancy when estrogenlevels are at their highest, large numbers of T-cellsutilize the DN pathway. As a consequence, theproduction of SP T-cells is at its nadir [15]. We esti-mate that during pregnancy only 2 % of total thymo-cytes reach the medulla.

(See figure on previous page.)Fig. 1 Proposed pathways for the production of T-cells in prepubertal mice. Progenitor cells enter the thymus via postcapillary venules (PCVs)located in the medulla and as T-cells leave by way of efferent lymphatic vessels (ELVs) located in the subcapsular cortex and in the medulla. Inprepubertal mice, the majority of thymocytes traverse the classic developmental pathway and as SP T-cells enter the lymphatic system (LS) (solidblack arrows) via ELVs located in the medulla. Lesser numbers of thymocytes enter the LS (dashed black arrows) as DN T-cells through ELVs locatedin the subscapular region

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 5 of 14

Fig. 2 (See legend on next page.)

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 6 of 14

DN T-cellsDN T-cells do not undergo positive selection (Figs. 1and 2). Consequently, they lack MHC restriction. Thisfactor, in combination with their unique TCR, producesbinding characteristics for γδ DN T-cells that differ sub-stantially from that of MHC restricted αβ T-cells. Wherethe latter bind to fragments (epitopes) of foreign antigenheld within the cleft of a class I or class II MHC mol-ecule [68], γδ DN T-cells do not. Instead, their bindingto foreign antigen is based on the conformational shapeof the intact antigen, similar to that of antibodies, andindependent of MHC involvement [69].There are three major subsets of γδ DN T-cells,

one of which is cytolytic. In humans this subset hasbeen characterized via its TCR as a Vγ9Vδ2 T-cell[69, 70]. When activated, they secrete interleukin-2(IL-2), interferon-γ (IFN-γ), and tumor necrosisfactor-β (TNF-β) [71]. These cytokines promote in-flammation, cytotoxicity, and delayed-type hypersensi-tivity (DTH) [72]. Vγ9Vδ2 T-cells are unconventionalin that non-proteins such as isoprenoids and alkyla-mines cause their activation [69]. Their venue of im-munological activity is in the peripheral bloodstream[70, 71]. Here they have an important role in bothtumor cell surveillance and anti-infective immunity[73]. The second subset of γδ DN T-cells has all thecharacteristics of the Vγ9Vδ2 T-cells, except they arenot cytolytic. The reason they are not is because theyhave an intermediate and incompletely expressedTCR/CD3 binding complex [74–76]. Henceforth theywill be referred to as γδ DN (int TCR/CD3) T-cells.Rather than being in the bloodstream, these T-cellsreside in the intraepithelial lymphocyte compartmentsof specific tissues such as skin, intestine, respiratorytract and uterus [69, 74]. The third subset of γδ DNT-cells are regulatory. In mice they are characterizedas Vγ6Vδ1 regulatory T-cells [77]. Activation of theseγδ DN regulatory T-cells results in the production of IL-10and transforming growth factor-β (TGF-β) [76, 78]. Thesecytokines control the action of cytotoxic T cells, NK cells,macrophages, dendritic cells and B cells [79]. The γδ DNregulatory T-cells are also restricted to the intraepitheliallymphocyte compartments of specific tissues [79]. In theuterus they play a significant role in the maintenance ofpregnancy.Alpha/beta DN T-cells are cytolytic [80] and produce

IL-4, IFN-γ, and TNF-β, but not IL-2 [81]. These T-cells

have a significant role in the control of intracellular bac-terial infection [82].

Immunomodulation, DN T-cells, and the maintenance ofpregnancyThe maintenance of pregnancy depends, to a large ex-tent, on the avoidance of maternal rejection. This isdealt with through the construction of an immunologicalbarrier using cells that lack the ability to express classicalHLA-A and HLA-B products [83]. This produces a pro-tective cocoon (trophoblast) in which MHC class I andMHC class II molecules are either missing or non-functional [84]; as a consequence, the processing andpresentation of antigens by MHC molecules cannot takeplace. SP T-cells are thus eliminated as a rejection factor,leaving only γδ DN T-cells to respond to the tropho-blast. Instead of rejection, however, these T-cells are es-sential for the maintenance of pregnancy. Thecomplexity of their overall role and the need for coord-ination requires extensive communication between γδDN T-cells and the decidua and trophoblast. Thetrophoblast, for example, initiates contact with a varietyof immune cells through its production and release ofchemokines. These are small proteins that act as ligandsto immune cell receptors. The binding of these uniqueligands to specific receptors results in the production ofadhesion molecules by respondent cells, thereby givingthem the means to adhere to the endothelium of bloodvessels. With this ability they are able to follow a chemo-kine concentration gradient to its source [85]. Cytokinesproduced by γδ DN T-cells, in contrast, encompass abroader application than chemokines in that they influencethe growth and receptivity of specific cell populations.The trophoblast attracts immune cells to the fetal-

maternal interface through its production of the che-mokines CXCL12 and CXCL16. For example, CXCL12recruits NK cells that have CXCR3 and CXCR4 re-ceptors [86, 87], and CXCL16 recruits αβ T-cells, γδDN T-cells, and monocytes through its interactionwith CXCR6 receptors [88]. Analyses of the deciduaduring early to mid-pregnancy has identified thepresence of the following cells: 1) γδ DN regulatoryT-cells; 2) γδ DN (int TCR/CD3) T-cells; 3) CD8+

cytotoxic T-cells; 4) CD4+ CD25+ Foxp3+ regulatoryT-cells; 5) NK cells; 6) dendritic cells; 7) macro-phages; and 8) neutrophils [75, 89, 90]. These cellshave all reached the decidua via the cardiovascular

(See figure on previous page.)Fig. 2 Proposed pathways for the production of T-cells in postpubertal mice. Progenitor cells enter the thymus via postcapillary venules (PCVs)located in the medulla and as T-cells leave by way of efferent lymphatic vessels (ELVs) located in the subcapsular cortex and in the medulla. Inpostpubertal mice, mast cell activation (red dots) results in large numbers of thymocytes exiting the classic pathway as DN T-cells and enteringthe LS (solid red arrows) via ELVs located in the subcapsular region. Lesser numbers of thymocytes remain in the classic pathway and enter the LS(dashed red arrows) as SP T-cells via ELVs located in the medulla

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 7 of 14

Fig. 3 Production of DN T-cells and SP T-cells by castrate adult animals. Shown are the percentages of DN T-cells and SP T-cells produced bycastrate adult animals. The numerical values were determined from the data of Pesic et al. [66]

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 8 of 14

Fig. 4 Production of DN T-cells and SP T-cells by intact adult animals. Shown are the percentages of DN T-cells and SP T-cells produced by intactadult animals. The numerical values were determined from the data of Pesic et al. [66]

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 9 of 14

system, with two exceptions. The exceptions are γδDN regulatory T-cells and γδ DN (int TCR/CD3) T-cells. These two subsets are part of a group that ob-tains access to their target tissues via the lymphaticsystem [69, 74, 75, 78, 91].In nonpregnant women, mice, rats, and rabbits, the

lymphatic system does not extend beyond the myome-trium [92–94]. Therefore, during early pregnancy, γδDN regulatory T-cells and γδ DN (int TCR/CD3) T-cellsare unable to respond to CXCL16 until lymphangiogen-esis (lymphatic vessel growth) has linked the endomet-rium to the lymphatic system. As a consequence, theseT-cells are the last to reach the fetal-maternal interface.Their late arrival indicates the likelihood that lymphan-giogenesis does not require their input, at least at thispoint. Their participation in the process comes later andis essential for the maintenance of pregnancy.Gamma/delta DN cytolytic T-cells are found in the

uterus during the early stages of pregnancy [95]. Theirpresence in this location is very likely due to CXCL16.However, the main function of these T-cells is to detectand destroy bacteria, and they are highly cytolytic. Thus,it is unusual for these cells to be in close proximity tothe trophoblast without causing its destruction [96]. Toprotect the trophoblast could be the reason why a largenumber of CD4+ CD25+ Foxp3+ regulatory T-cells residein the decidua [89]. These regulatory T-cells are fullycapable of eliminating γδ DN cytolytic T-cells [97, 98]. Itis noteworthy that Foxp3+ regulatory T-cells are amongthe first immune cells to enter the uterus, indicating thatthey are in place prior to the entry of the γδ DNcytolytic T-cells [99]. Levels of Foxp3+ regulatory T-cells undergo a significant increase during pregnancy[5, 89, 100, 101], with the decidua being the majorrecipient of their enhanced production [89]. It shouldbe noted that this form of protection for the tropho-blast has an upper limit since excess numbers of per-ipheral γδ DN cytolytic T-cells can cause abortion[96, 102, 103]. It was not reported in these studies ifthe increase in γδ DN cytolytic T-cells was due toacute bacterial infection [104, 105]. Putative evidenceof the involvement of Foxp3+ regulatory T-cells inpreventing abortion is indicated by reports thatwomen with decreased levels of these T-cells sufferfrom recurrent miscarriages [106–108].NK cells play a significant role in the creation of blood

and lymphatic vessels. Their major responsibility is toproduce a large number of cytokines. These include vas-cular endothelial growth factor (VEGF), fibroblastgrowth factor (FGF), TNF-β, IFN-γ, and the angiopoie-tins, to name a few [109]. Blood-borne NK cells arecytolytic and fully capable of destroying the trophoblast[110]. However, unlike the γδ DN cytolytic T-cells, theyare not eliminated. Instead, they are converted into

noncytolytic NK cells. This transformation is under thecontrol of TGF-β, and involves converting cytolyticCD56dim CD16+ peripheral NK cells (CD16+ pNK cells)into noncytolytic CD56bright CD16— uterine NK cells(CD16— uNK cells) [111–113]. The initial source ofTGF-β for pNK cell conversion is provided by the male,and TGF-β reaches the decidual area via the ejaculate[114–116]. TGF-β is also produced by decidual stromalcells [112]. However, the overall supply of TGF-β is notinexhaustible. The TGF-β derived from the ejaculate islimited for obvious reasons, and the ability of stromalcells to produce the cytokine is seriously compromised.This is because TGF-β is involved in two simultaneousand conflicting operations. In addition to convertingpNK cells into uNK cells, TGF-β is also involved in im-plantation. Its role in this process is to initiate the apop-totic destruction of decidual stromal cells.Shooner et al. [117] noted that stromal cells of the

pregnant rat uterus undergo a TGF-β-induced increasein apoptosis between day 5 and day 14 of pregnancy.During this period, the loss in stromal cells is correlatedwith decreased production of the two isoforms, TGF-β1and TGF-β2. After day 14, only limited quantities ofTGF-β are produced by stromal cell survivors. Withoutreplenishment, the decrease in TGF-β could have a ser-ious impact on the transformation of pNK cells to uNKcells. Red-Horse [94] noted that lymphatic vessels in theendometrial area of pregnant mice begin their develop-ment between embryonic day 9.0 and day 9.5. Thiswould indicate that these lymphatic vessels have ~ 5 daysto complete their development before TGF-β is seriouslydepleted. This timeframe is critical since γδ DN regula-tory T-cells, a major source of TGF-β, can only reachthe fetal-maternal interface via the newly-formed lymph-atic vessels.TGF-β is regarded as a pleiotropic cytokine. This

characteristic is obvious during the maintenance ofpregnancy. Here, the cytokine has a significant impacton lymphangiogenesis by controlling levels of pNKcells [112]. However, while TGF-α is performing thisfunction it is undergoing self-destruction by initiatingthe apoptosis of decidual stromal cells [117]. Bothprocesses are essential for the maintenance of preg-nancy. The prospect of the cytokine being depletedduring implantation is troublesome. One couldvisualize scenarios in which the levels of stromal cellswere lower than normal, or where TGF-β-inducedstromal cell apoptosis occurred at a faster rate. Inthese instances, implantation would be successful,whereas a scarcity of TGF-β could alter the formationof lymphatic vessels. If this occurred, it would preventγδ DN regulatory T-cells from reaching the fetal-maternal interface. The loss of a major source ofTGF-β could impede the conversion of pNK cells to

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 10 of 14

uNK cells. Notably, a number of studies have re-ported that excess levels of pNK in pregnant womenare highly correlated with recurrent spontaneousabortion [118–125].

ConclusionsIn this review we have presented evidence indicatingthat sex steroids initiate a pathway for the production ofDN T-cells. This is done through the mediation ofthymic mast cells. In females, the DN pathway is con-trolled by estrogen. During pregnancy, estrogen levelsincrease [126], causing the production of DN T-cells totake priority over the production of SP T-cells. Thisguarantees that the trophoblast will have a plentiful andever increasing supply of γδ DN regulatory T-cells andγδ DN (int TCR/CD3) T-cells. In addition to their in-creased production, these two T-cell subsets are specific-ally guided to the trophoblast by CXCL16; and, aftertravelling through efferent lymphatic vessels and newly-formed endometrial lymphatic vessels, they performtheir role in the maintenance of pregnancy. This role in-volves the production of cytokines; a process that re-quires T-cell activation. Since γδ DN T-cells and thetrophoblast both lack MHC restriction, this activationoccurs by binding to an intact antigen. Heyborne et al.have proposed that heat shock protein-60 (HSP-60) hasall the attributes to be that antigen [127].Activation of γδ DN (int TCR/CD3) T-cells results in

the production of TNF-α and IFN-γ. These cytokinesare responsible for maintaining the integrity of the bloodand lymphatic vessels [128–130]. As important as this isto maintaining pregnancy, activation of γδ DN regula-tory T-cells, and their production of IL-10 and TGF-β, isof far greater consequence. IL-10 by itself promotestrophoblast invasion, and suppresses trophoblast apop-tosis [78]; whereas TGF-β is involved in lymphangiogen-esis and implantation [112, 117]. However, when IL-10and TGF-β act in combination, the two cytokines per-form a synergistic suppression of the cytolytic activitiesof γδ DN cytolytic T-cells and pNK cells [131]. Thiswould strongly suggest that the two cytokines are sup-planting, or adding to, the role of the CD4+ CD25+

Foxp3+ regulatory T-cells in protecting the trophoblast.The importance of γδ DN regulatory T-cells in themaintenance of pregnancy has been demonstrated byArck et al. [96]. This group found that the rate of abor-tion in pregnant mice underwent a significant increasewhen the animals were given monoclonal antibodiesagainst γδ DN regulatory T-cells. Notably, these micewere injected with the antibodies after 8.5 days of gesta-tion, coincident with the time when γδ DN regulatoryT-cells first reach the decidua, and 3.5 days after im-plantation. One could speculate that construction ofendometrial lymphatic vessels was the reason that γδ

DN regulatory T-cells did not reach the decidua prior to8.5 days of gestation.The production of DN T-cells in anti-infective im-

munity can take place without sex steroid involvment.For example, in response to the direct invasion ofbacteria and tumor cells [132–134], mast cell activa-tion occurs when the Fc component of either IgG orIgE antibodies bind to FcγR and FcεR receptors. Thetwo receptors are also located on the membrane ofmast cells, and the immunoglobulins are the productof an antigen-induced activation of B cells and theirsubsequent differentiation into antibody-secretingplasma cells. With this brief description of theantibody-induced initiation of the DN T-cell pathwaywe conclude this review. We should point out thatthis is only one of the ways that γδ DN T-cells act asearly sensors of stress and infection. Admittedly,much of this review has been about the role of γδDN T-cells in reproduction. However, in doing thiswe have presented a careful overview of current re-search on sex steroid-induced “thymocyte loss.” Welook forward to future research to advance our under-standing of the role of γδ DN T-cells in reproductionas well as in immunology.

AbbreviationsELVs: Efferent lymphatic vessels; LVs: Lymphatic vessels; PCVs: Postcapillaryvenules; DN T-cells: CD4— CD8— T-cells; SP T-cells: CD4+ CD8— T-cells andCD4—CD8+ T-cells.

Competing interestsThe authors declare that they have no competing interests.

Authors’ contributionsAll authors have contributed to the writing of this review, and all have readand approved the final manuscript.

Received: 25 February 2015 Accepted: 8 July 2015

References1. Albrecht ED, Aberdeen GW, Pepe GJ. The role of estrogen in the

maintenance of primate pregnancy. Am J Obstet Gynecol. 2000;182:432–8.2. Olsen NJ, Kovacs WJ. Gonadal steroids and immunity. Endocr Rev.

1996;17:369–84.3. Olsen NJ, Olson G, Viselli SM, Gu X, Kovacs WJ. Androgen receptors in

thymic epithelium modulate thymus size and thymocyte development.Endocrinology. 2001;142:1278–83.

4. Kuhl H, Gross M, Schneider M, Weber W, Mehlis W, Stegmuller M, et al.The effect of sex steroids and hormonal contraceptives upon thymus andspleen on intact female rats. Contraception. 1983;28:587–601.

5. Zoller AL, Schnell FJ, Kersh GJ. Murine pregnancy leads to reducedproliferation of maternal thymocytes and decreased thymic emigration.Immunology. 2007;121:207–15.

6. Hince M, Sakkal S, Vlahos K, Dudakov J, Boyd R, Chidgey A. The role of sexsteroids and gonadectomy in the control of thymic involution. CellImmunol. 2008;252:122–38.

7. Zaitsu M, Narita S, Lambert KC, Grady JJ, Estes DM, Curran EM, et al.Estradiol activates mast cells via a non- genomic estrogen receptor-α andcalcium influx. Mol Immunol. 2007;44:1977–85.

8. Metcalfe DD. Mast cells and mastocytosis. Blood. 2008;112:946–56.9. Maruyama S, Tsukahara A, Suzuki S, Tada T, Minagawa M, Watanabe H, et al.

Quick recovery in the generation of self-reactive CD4low natural killer (NK) T

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 11 of 14

cells by an alternative pathway when restored from acute thymic atrophy.Clin Exp Immunol. 1999;117:587–95.

10. Chmeilewski V, Drupt F, Morfin R. Dexamethasone induced apoptosis ofmouse thymocytes: prevention by native 7α-hydroxysteroids. Immunol CellBiol. 2000;78:238–46.

11. Yao G, Hou Y. Thymic atrophy via estrogen-induced apoptosis is related toFas/FasL pathway. Int Immunopharmacol. 2004;4:213–21.

12. Okasha SA, Ryu S, Do Y, McKallip RJ, Nagarkatti M, Nagarkatti PS. Evidencefor estradiol-induced apoptosis and dysregulated T cell maturation in thethymus. Toxicology. 2001;163:49–62.

13. Do Y, Ryu S, Nagarkatti M, Nagarkatti PS. Role of death receptorpathway in estradiol-induced T-cell apoptosis in vivo. Toxicol Sci.2002;70:63–72.

14. Staples JE, Fiore NC, Frazier Jr DE, Gasiewicz TA, Silverstone AE.Overexpression of the anti-apoptotic oncogene, bcl-2, in the thymus does notprevent atrophy induced by estradiol or 2,3,7,8-tetrachlorodibenzo-p-dioxin.Toxicol Appl Pharmacol. 1998;151:200–10.

15. Zoller AL, Kersh GJ. Estrogen induces thymic atrophy by eliminating earlythymic progenitors and inhibiting proliferation of β-selected thymocytes.J Immunol. 2006;176:7371–8.

16. Bebo Jr BF, Fyfe-Johnson A, Adlard K, Beam AG, Vandenbark AA, Offner H.Low-dose estrogen therapy ameliorates experimental autoimmuneencephalomyelitis in two different inbred mouse strains. J Immunol.2001;166:2080–9.

17. Rijhsinghani AG, Thompson K, Bhatia SK, Waldschmidt TJ. Estrogen blocksearly T cell development in the thymus. Am J Reprod Immunol.1996;36:269–77.

18. Martin A, Alonso LM, Gomez del Moral M, Zapata AG. Ultrastructuralchanges in the adult rat thymus after estradiol benzoate treatment.Tissue Cell. 1994;26:169–79.

19. Martin A, Alonso L, Gomez del Moral M, Zapata AG. Morphometricalchanges in the rat thymic lymphoid cells after treatment with two differentdoses of estradiol benzoate. Histol Histopath. 1994;9:281–6.

20. Kato S. Intralobular lymphatic vessels and their relationship to blood vesselsin the mouse thymus. Light- and electron-microscopic study. Cell TissueRes. 1988;253:181–7.

21. Kato S. Thymic microvascular system. Microsc Res Tech. 1997;38:287–99.22. Weiss L. Electron microscopic, observations on the vascular barrier in the

cortex of the thymus of the mouse. Anat Rec. 1963;145:413–37.23. Pearse G. Normal structure, function and histology of the thymus. Toxicol

Pathol. 2006;34:504–14.24. Oner H, Ozan E. Effects of gonadal hormones on thymus gland after

bilateral ovariectomy and orchidectomy in rats. Arch Androl.2002;48:115–26.

25. Okuyama R, Abo T, Seki S, Ohteki T, Sugiura K, Kusumi A, et al. Estrogenadministration activates extrathymic T cell differentiation in the liver.J Exp Med. 1992;175:661–9.

26. Kimura M, Hanawa H, Watanabe H, Ogawa M, Abo T. Synchronousexpansion of intermediate TCR cells in the liver and uterus duringpregnancy. Cell Immunol. 1995;162:16–25.

27. Pardoll DM, Fowlkes BJ, Lew AM, Maloy WL, Weston MA, Bluestone JA, et al.Thymus-dependent and thymus-independent developmental pathways forperipheral T cell receptor-γδ-bearing lymphocytes. J Immunol.1988;140:4091–96.

28. Boyd RL, Tucek CL, Godfrey DI, Izon DJ, Wilson TJ, Davidson NJ, et al. Thethymic microenvironment. Immunol Today. 1993;14:445–59.

29. Kendall MD. Functional anatomy of the thymic microenvironment. J Anat.1991;177:1–29.

30. Egerton M, Scollay R, Shortman K. Kinetics of mature T-cell development inthe thymus. Proc Natl Acad Sci U S A. 1990;87:2579–82.

31. Odaka C, Morisada T, Oike Y, Suda T. Distribution of lymphatic vessels inmouse thymus: immunofluorescence analysis. Cell Tissue Res.2006;325:13–22.

32. Yong LC, Watkins SG, Boland JE. The mast cell: III. Distribution andmaturation in various organs of the young rat. Pathology. 1979;11:427–45.

33. Raica M, Cimpean AM, Nico B, Guidolin D, Ribatti D. A comparativestudy of the spatial distribution of mast cells and microvessels in thefoetal and adult thymus and thymoma. Int J Exp Pathol. 2010;91:17–23.

34. Ruitenberg EJ, Buys J. Thymus atrophy during early pregnancy and its effecton a Trichinella spiralis infection in mice, including intestinal pathology andblood eosinophilia. Vet Immun Immunopath. 1980;1:199–214.

35. Chatamra K, Daniel PM, Kendall MD, Lam DK. Atrophy of the thymus in ratsrendered diabetic by strepozotocin. Horm Metab Res. 1985;17:630–2.

36. Majno G, Palade GE, Schoefl GI. Studies on inflammation. II. The site ofaction of histamine and serotonin along the vascular tree: a topographicstudy. J Biophys Biochem Cytol. 1961;11:607–26.

37. Schoefl GI. The migration of lymphocytes across the vascular endotheliumin lymphoid tissue. A reexamination. J Exper Med. 1972;136:568–88.

38. Savagner P, Imhof BA, Yamada KM, Thiery J-P. Homing of hemopoieticprecursor cells to the embryonic thymus: characterization of an invasivemechanism induced by chemotactic peptides. J Cell Biol.1986;103:2715–27.

39. Dunon D, Imhof BA. Mechanisms of thymus homing. Blood. 1993;81:1–8.40. Petri B, Bixel MG. Molecular events during leukocyte diapedesis. FEBS J.

2006;273:4399–407.41. Farr AG, De Bruyn PP. The mode of lymphocyte migration through

postcapillary venule endothelium in lymph node. Amer J Anat.1975;143:59–92.

42. Williams RM, Chanana AD, Cronkite EP, Waksman BH. Antigenic markers oncells leaving calf thymus by way of the efferent lymph and venous blood.J Immunol. 1971;106:1143–46.

43. Chanana AD, Cronkite EP, Joel DD, Williams RM, Waksman BH. Migration ofthymic lymphocytes: immunofluorescence and 3HTdR labeling studies.Adv Exp Med Biol. 1971;12:113–18.

44. Kotani M, Seiki K, Yamashita A, Horii I. Lymphatic drainage of thymocytes tothe circulation in the guinea pig. Blood. 1966;27:511–20.

45. Ushiki T. A scanning electron-microscopic study of the rat thymus withspecial reference to cell types and migration of lymphocytes into thegeneral circulation. Cell Tissue Res. 1986;244:285–98.

46. Miyasaka M, Pabst R, Dudler L, Cooper M, Yamaguchi K. Characterization oflymphatic and venous emigrants from the thymus. Thymus. 1990;16:29–43.

47. Azzali G, Orlandini G, Gatti R. The migration of lymphocytes andpolymorphonuclear leukocytes across the endothelial wall of the absorbingperipheral lymphatic vessel. J Submicrosc Cytol Pathol. 1990;22:543–49.

48. Baluk P, Fuxe J, Hashizume H, Romano T, Lashnits E, Butz S, et al.Functionally specialized junctions between endothelial cells of lymphaticvessels. J Exp Med. 2007;204:2349–62.

49. Lind EF, Prockop SE, Porritt HE, Petrie HT. Mapping precursor movementthrough the postnatal thymus reveals specific microenvironmentssupporting defined stages of early lymphoid development. J Exp Med.2001;194:127–34.

50. Takahama Y. Journey through the thymus: stromal guides for T-celldevelopment and selection. Nat Rev Immunol. 2006;6:127–35.

51. Wilson A, Capone M, MacDonald RM. Unexpectedly late expression ofintracellular CD3ε and TCR γδ proteins during adult thymus development.Int Immunol. 1999;11:1641–50.

52. Watanabe H, Seki S, Sugiura K, Hirokawa K, Utsuyama M, Takahashi-IwanagaH, et al. Ontogeny and development of extrathymic T cells in liver.Immunology. 1992;77:556–63.

53. Abo T. Extrathymic pathways of T-cell differentiation and immunomodulation.Int Immunopharmacol. 2001;1:1261–73.

54. Narita S, Goldblum RM, Watson CS, Brooks EG, Estes DM, Curran EM, et al.Environmental estrogens induce mast cell degranulation and enhanceIgE-mediated release of allergic mediators. Environ Health Perspect.2007;115:48–52.

55. Mobbs CV, Kannegieter LS, Finch CE. Delayed anovulatory syndromeinduced by estradiol in female C57BL/6 J mice: age-like neuroendocrine,but not ovarian impairments. Biol Reprod. 1985;32:1010–17.

56. Webb SJ, Geoghegan TE, Prough RA, Michael Miller KK. The biologicalactions of dehydroepiandrosterone involves multiple receptors. Drug MetabRev. 2006;38:89–116.

57. Parker Jr CR, Conway-Meyers BA. The effects of dehydroepiandrosterone(DHEA) on the thymus, spleen, and adrenals of prepubertal and adultfemale rats. Endocr Res. 1998;24:113–26.

58. Wilhelm M, King B, Silverman AJ, Silver R. Gonadal steroids regulate thenumber and activational state of mast cells in the medial habenula.Endocrinology. 2000;141:1178–86.

59. Popova LA. Effect of ovarian hormones on mast cells in the denervateduterus of rats. Fiziol Zh. 1989;35:75–80.

60. Bartke A, Steele RE, Musto N, Caldwell BV. Fluctuations in plasmatestosterone levels in adult male rats and mice. Endocrinology.1973;92:1223–28.

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 12 of 14

61. Chapman JC, Min S, Kunaporn S, Tung K, Shah S, Michael SD.The administration of cortisone to female B6A mice during their immuneadaptive period causes anovulation and the formation of ovarian cysts.Am J Reprod Immunol. 2002;48:184–9.

62. Mitak M, Gojmerac T, Mandic B, Cvetnic Z. Changes in serum concentrationsof 17β-estradiol in female rats during estrous cycle after treatment withatrazine and zearalenone. Vet Med Czech. 2001;5:145–8.

63. Lindberg MK, Weihua Z, Andersson N, Moverare S, Gao H, Vidal O, et al.Estrogen receptor specificity for the effects of estrogen in ovariectomizedmice. J Endocrinol. 2002;174:167–78.

64. Staples JE, Gasiewicz TA, Fiore NC, Lubahn DB, Korach KS, Silverstone AE.Estrogen receptor α is necessary in thymic development andestradiol-induced thymic alterations. J Immunol. 1999;163:4168–74.

65. Anderson MS, Venanzi ES, Klein L, Chen Z, Berzins SP, Turley SJ, et al.Projection of an immunological self shadow within the thymus by the aireprotein. Science. 2002;298:1385–401.

66. Pesic V, Radojevic K, Kosec D, Plecas-Sloarovic B, Perisic M, Leposavic G.Peripubertal orchidectomy transitorily affects age-associated thymicinvolution in rats. Braz J Med Biol Res. 2007;40:1481–93.

67. Naquet P, Naspetti M, Boyd R. Development, organization and function ofthe thymic medulla in normal, immunodeficient or autoimmune mice.Semin Immunol. 1999;11:47–55.

68. Rotzschke O, Falk K, Deres K, Schild H, Norda M, Metzger J, et al. Isolationand analysis of naturally processed viral peptides as recognized by cytotoxicT cells. Nature. 1990;348:252–4.

69. Girardi M. Immunosurveillance and immunoregulation of γδ T cells. J InvestDermatol. 2006;126:25–31.

70. Takihara Y, Reimann J, Michalopoulos E, Ciccone E, Moretta L, Mak TW.Diversity and structure of human T cell receptor delta chain genes inperipheral blood gamma/delta-bearing T lymphocytes. J Exp Med.1989;169:393–405.

71. Barakonyi A, Miko E, Varga P, Szekeres-Bartho J. V-chain preference ofgamma/delta T-cell receptors in peripheral blood during term labor. Am JReprod Immunol. 2008;59:201–5.

72. Cameron MJ, Kelvin DJ. Cytokines and chemokines-their receptors and theirgenes: an overview. Adv Exp Med Biol. 2003;520:8–32.

73. Kabelitz D, Peters C, Wesch D, Oberg HH. Regulatory functions of γδ T cells.Int Immunopharmacol. 2013;16:382–7.

74. Ebert EC, Roberts AI, Brolin RE, Raska K. Examination of the low proliferativecapacity of human jejunal intraepithelial lymphocytes. Clin Exp Immunol.1986;65:148–57.

75. Mincheva-Nilsson L, Hammarström S, Hammarström ML. Human decidualleukocytes from early pregnancy contain high numbers of gammadelta + cells and show selective down-regulation of alloreactivity. JImmunol. 1992;149:2203–11.

76. Nagaeva O, Jonsson L, Mincheva-Nilsson L. Dominant IL-10 and TGF-betamRNA expression in gamma/delta T-cells of human early pregnancydecidua suggests immunoregulatory potential. Am J Reprod Immunol.2002;48:9–17.

77. Heyborne KD, Cranfill RL, Carding SR, Born WK, O'Brien RL. Characterizationof γδ T lymphocytes at the maternal-fetal interface. J Immunol.1992;149:2872–8.

78. Fan D-X, Duan J, Li M-Q, Xu B, Li D-J, Jin L-P. The decidual gamma-delta Tcells up-regulate the biological functions of trophoblasts via IL-10 secretionin early human pregnancy. Clin Immunol. 2011;141:284–92.

79. Groux H. An overview of regulatory T cells. Microbes Infect. 2001;3:883–9.80. Ballas ZK, Rasmussen W. NK1.1+ thymocytes. Adult murine CD4-,

CD8- thymocytes contain an NK1.1+, CD3+, CD5hi, CD44hi, TCR-Vbeta 8+ subset. J Immunol. 1990;145:1039–45.

81. Zlotnik A, Godfrey DI, Fischer M, Suda T. Cytokine production by matureand immature CD4-CD8- T cells. Alpha beta-T cell receptor + CD4-CD8- Tcells produce IL-4. J Immunol. 1992;149:1211–15.

82. Cowley SC, Hamilton E, Frelinger JA, Su J, Forman J, Elkins KL. CD4-CD8- Tcells control intracellular bacterial infections both in vitro and in vivo. J ExpMed. 2005;202:309–19.

83. Geirsson A, Paliwal I, Lynch RJ, Bothwell AL, Hammond GL. Class IItransactivator promoter activity is suppressed through regulation by atrophoblast noncoding RNA. Transplantation. 2003;76:387–94.

84. Barakonyi A, Kovacs KT, Miko E, Szereday L, Varga P, Szekeres-Bartho J.Recognition of nonclassical HLA class I antigens by gamma delta T cellsduring pregnancy. J Immunol. 2002;168:2683–8.

85. Park D-W, Yang K-M. Hormonal regulation of uterine chemokines andimmune cells. Clin Exp Reprod Med. 2011;38:179–85.

86. Wu X, Jin LP, Yuan MM, Zhu Y, Wang MY, Li DJ. Human first-trimestertrophoblast cells recruit CD56brightCD16— NK cells into decidua by way ofexpressing and secreting of CXCL12/stromal cell-derived factor 1. JImmunol. 2005;175:61–8.

87. Hanna J, Wald O, Goldman-Wohl D, Prus D, Markel G, Gazit R, et al. CXCL12expression by invasive trophoblasts induces the specific migration ofCD16— human natural killer cells. Blood. 2003;102:1569–77.

88. Huang Y, Zhu XY, Du MR, Li DJ. Human trophoblasts recruited Tlymphocytes and monocytes into deciduas by secretion of CXCL16 andinteraction with CXCR6 in the first-trimester pregnancy. J Immunol.2008;180:2367–75.

89. Dimova T, Nagaeva O, Stenqvist A-C, Hedlund M, Kjellberg L, Strand M, et al.Maternal foxp3 expressing CD4+ CD25+ and CD4+ CD25—regulatory T-cellpopulations are enriched in human early normal pregnancy decidua: aphenotypic study of paired decidual and peripheral blood samples. Am JReprod Immunol. 2011;66 Suppl 1:44–56.

90. Blois SM, Kammerer U, Alba Soto C, Tometten MC, Shaikly V, Barrientos G,et al. Dendritic cells: key to fetal tolerance? Biol Reprod. 2007;77:590–8.

91. Maroni ES, de Sousa MA. The lymphoid organs during pregnancy in themouse. A comparison between syngeneic and allogeneic mating. Clin ExpImmunol. 1973;13:107–24.

92. Gray H. Anatomy of the human body. Philadelphia: Lea and Febiger; 1985.93. Koukourakis MI, Giatromanolaki A, Sivridis E, Simopoulos C, Gatter KC, Harris

AL, et al. LYVE-1 immunohistochemical assessment of lymphangiogenesis inendometrial and lung cancer. J Clin Pathol. 2005;58:202–6.

94. Red-Horse K. Lymphatic vessel dynamics in the uterine wall. Placenta.2008;29(Suppl A):S55–9.

95. Arck PC, Ferrick DA, Steele-Norwood D, Egan PJ, Croitoru K, Carding SR,et al. Murine T cell determination of pregnancy outcome. Cell Immunol.1999;196:71–9.

96. Arck PC, Ferrick DA, Steele-Norwood D, Croitoru K, Clark DA. Murine Tcell determination of pregnancy outcome: I. effects of strain, alpha/betaT cell receptor, gamma/delta T cell receptor, and gamma/delta T cellsubsets. Am J Reprod Immunol. 1997;37:492–502.

97. Goncalves-Sousa N, Ribot JC, de Barros A, Correia DV, Caramalho I,Silva-Santos B. Inhibition of murine gammadelta lymphocyte expansionand effector function by regulatory alphabeta T cells is cell-contact-dependent and sensitive to GITR modulation. Eur J Immunol.2010;40:61–70.

98. Mahan CS, Thomas JJ, Boom WH, Rojas RE. CD4+ CD25high Foxp3+

regulatory T cells downregulate human Vδ2+ T-lymphocyte functiontriggered by anti-CD3 or phosphoantigen. Immunology. 2009;127:398–407.

99. Kallikourdis M, Betz AG. Periodic Accumulation of Regulatory T Cells in theUterus: Preparation for the Implantation of a Semi-Allogeneic Fetus? PLoSOne. 2007;2, e382.

100. Aluvihare VR, Kallikourdis M, Betz AG. Regulatory T cells mediate maternaltolerance to the fetus. Nat Immunol. 2004;5:266–71.

101. Somerset DA, Zheng Y, Kilby MD, Sansom DM, Drayson MT. Normal humanpregnancy is associated with an elevation in the immune suppressive CD25+ CD4+ regulatory T-cell subset. Immunology. 2004;112:38–43.

102. Barakonyi A, Polgar B, Szekeres-Bartho J. The role of gamma/delta T-cellreceptor-positive cells in pregnancy: part II. Am J Reprod Immunol.1999;42:83–7.

103. Szekeres-Bartho J, Barakonyi A, Polgar B, Par G, Faust Z, Palkovics T, et al.The role of γδ T cells in progesterone-mediated immunomodulation duringpregnancy: a review. Am J Reprod Immunol. 1999;42:44–8.

104. Hara T, Mizuno Y, Takaki K, Akeda H, Aoki T, Nagata M, et al. Predominantactivation and expansion of Vγ9-bearing γδ T cells in vivo as well as in vitroin Salmonella infection. J Clin Invest. 1992;90:204–10.

105. Munk ME, Gatrill AJ, Kaufmann SH. Target cell lysis and IL-2 secretion by γδT lymphocytes after activation with bacteria. J Immunol. 1990;145:2434–9.

106. Du MR, Guo PF, Piao HL, Wang SC, Sun C, Jin LP, et al. Embryonictrophoblasts induce decidual regulatory T cell differentiation andmaternal–fetal tolerance through thymic stromal lymphopoietin instructingdendritic cells. J Immunol. 2014;192:1502–11.

107. Sasaki Y, Sakai M, Miyazaki S, Higuma S, Shiozaki A, Saito S. Decidual andperipheral blood CD4+ CD25+ regulatory T cells in early pregnancy subjectsand spontaneous abortion cases. Mol Hum Reprod. 2004;10:347–53.

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 13 of 14

108. Winger EE, Reed JL. Low circulating CD4(+) CD25(+) Foxp3(+) T regulatorycell levels predict miscarriage risk in newly pregnant women with a historyof failure. Am J Reprod Immunol. 2011;66:320–28.

109. Zygmunt M, Herr F, Münstedt K, Lang U, Liang OD. Angiogenesis andvasculogenesis in pregnancy. Eur J Obstet Gynecol Reprod Biol. 2003;110Suppl 1:S10–8.

110. Olivares EG, Munoz R, Tejerizo G, Montes MJ, Gomez-Molina F,Abadia-Molina AC. Decidual lymphocytes of human spontaneousabortions induce apoptosis but not necrosis in JEG-3 extravilloustrophoblast cells. Biol Reprod. 2002;67:1211–7.

111. King A, Jokhi PP, Burrows TD, Gardner L, Sharkey AM, Loke YW. Functions ofhuman decidual NK cells. Am J Reprod Immunol. 1996;35:258–60.

112. Keskin DB, Allan DS, Rybalov B, Andzelm MM, Stern JN, Kopcow HD, et al.TGF beta promotes conversion of CD16+ peripheral blood NK cells intoCD16— NK cells with similarities to decidual NK cells. Proc Natl Acad Sci U SA. 2007;104:3378–83.

113. Kopcow HD, Allan DS, Chen X, Rybalov B, Andzelm MM, Ge B, et al. Humandecidual NK cells form immature activating synapses and are not cytotoxic.Proc Natl Acad Sci U S A. 2005;102:15563–8.

114. Sharkey DJ, Macpherson AM, Tremellen KP, Mottershead DG, Gilchrist RB,Robertson SA. TGF-β mediates proinflammatory seminal fluid signaling inhuman cervical epithelial cells. J Immunol. 2012;189:1024–35.

115. Nocera M, Chu TM. Characterization of latent transforming growth factor-betafrom human seminal plasma. Am J Reprod Immunol. 1995;33:282–91.

116. Tremellen KP, Seamark RF, Robertson SA. Seminal Transforming GrowthFactor 1, Stimulates Granulocyte-Macrophage Colony-Stimulating FactorProduction and Inflammatory Cell Recruitment in the Murine Uterus. BiolReprod. 1998;58:1217–25.

117. Shooner C, Caron PL, Fréchette-Frigon G, Leblanc V, Déry MC, Asselin E.TGF-beta expression during rat pregnancy and activity on decidual cellsurvival. Reprod Biol Endocrin. 2005;3:20–38.

118. Aoki K, Kajiura S, Matsumoto Y, Oqasawara M, Okada S, Yaqami Y, et al.Preconceptional natural-killer-cell activity as a predictor of miscarriage.Lancet. 1995;345:1340–2.

119. Higuchi K, Aoki K, Kimbara T, Hosoi N, Yamamoto T, Okada H. Suppressionof natural killer cell activity by monocytes following immunotherapy forrecurrent spontaneous aborters. Am J Reprod Immunol. 1995;33:221–7.

120. Lachapelle MH, Miron P, Hemmings R, Roy DC. Endometrial T, B, and NKcells in patients with recurrent spontaneous abortion. Altered profile andpregnancy outcome. J Immunol. 1996;156:4027–34.

121. Clifford K, Flanagan AM, Regan L. Endometrial CD56+ natural killer cells inwomen with recurrent miscarriage: a histomorphometric study. HumReprod. 1999;14:2727–30.

122. Emmer PM, Nelen WL, Steegers EA, Hendriks JC, Veerhoek M, Joosten I.Peripheral natural killer cytotoxicity and CD56+ CD16+ cells increase duringearly pregnancy in women with a history of recurrent spontaneousabortion. Hum Reprod. 2000;15:1163–9.

123. Park DW, Lee HJ, Park CW, Hong SR, Kwak-Kim J, Yang KM. Peripheral bloodNK cells reflect changes in decidual NK cells in women with recurrentmiscarriages. Am J Reprod Immunol. 2010;63:173–80.

124. Junovich G, Azpiroz A, Incera E, Ferrer C, Pasqualini A, Gutierrez G.Endometrial CD16(+) and CD16(−) NK cell count in fertility and unexplainedinfertility. Am J Reprod Immunol. 2013;70:182–9.

125. Seshadri S, Sunkara SK. Natural killer cells in female infertility and recurrentmiscarriage: a systematic review and meta-analysis. Hum Reprod Update.2014;20:429–38.

126. Abbassi-Ghanavati M, Greer LG, Cunningham FG. Pregnancy and laboratorystudies: a reference table for clinicians. Obstet Gynecol. 2009;114:1326–31.

127. Heyborne K, Fu YX, Nelson A, Farr A, O'Brien R, Born W. Recognition oftrophoblasts by gamma delta T cells. J Immunol. 1994;153:2918–26.

128. Ashkar AA, Di Santo JP, Croy BA. Interferon gamma contributes to initiationof uterine vascular modification, decidual integrity, and uterine naturalkiller cell maturation during normal murine pregnancy. J Exp Med.2000;192:259–70.

129. Murphy SP, Tayade C, Ashkar AA, Hatta K, Zhang J, Croy BA. Interferongamma in successful pregnancies. Biol Reprod. 2009;80:848–59.

130. Folkman J, Klagsbrun M. Angiogenic factors. Science. 1987;235:442–47.131. Seo N, Tokura Y, Takigawa M, Egawa K. Depletion of IL-10 and TGF-β-

producing γδ T cells by administering a daunomycin-conjugated specificmonoclonal antibody in early tumor lesions augments the activity of CTLsand NK cells. J Immunol. 1999;163:242–49.

132. Abo T, Sugawara S, Seki S, Fujii M, Rikiishi H, Takeda K, et al. Induction ofhuman TCR gamma delta + and TCR gamma delta-CD2 + CD3- doublenegative lymphocytes by bacterial stimulation. Int Immunol. 1990;2:775–85.

133. Abo T, Kusumi A, Seki S, Ohteki T, Sugiura K, Masuda T, et al. Activation ofextrathymic T cells in the liver and reciprocal inactivation of intrathymic Tcells by bacterial stimulation. Cell Immunol. 1992;142:125–36.

134. Seki S, Abo T, Sugiura K, Ohteki T, Kobata T, Yagita H, et al. Reciprocal T cellresponses in the liver and thymus of mice injected with syngeneic tumorcells. Cell Immunol. 1991;137:46–60.

Submit your next manuscript to BioMed Centraland take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit

Chapman et al. Reproductive Biology and Endocrinology (2015) 13:73 Page 14 of 14